Aminoacyl-tRNA synthetases (ARSs) establish the rules of the genetic code, whereby each amino acid (aa) is attached to a cognate tRNA. Errors in this process lead to mistranslation, which can be toxic to cells. Recent studies suggest that the selective forces exerted by cell-specific requirements and environmental conditions potentially shape quality control mechanisms. Approximately half of the ARSs possess a proofreading function to hydrolyze mischarged aa-tRNAs and evidence that non-protein aa metabolites pose the greatest threat to fidelity is beginning to emerge. Interestingly, single-domain proteins homologous to ARS editing domains are encoded in many genomes but many open questions regarding the physiological function of these putative trans-editing proteins remain. While proofreading of genetically-encoded aa's by ARSs is well documented, much less is known about how the translation quality control machinery prevents misincorporation of non- protein aa's. We hypothesize that tRNA-specific, as well as semi-promiscuous trans-editing proteins, which are capable of acting on a variety of tRNA substrates and whose expression can be regulated independent of the ARSs, have evolved to prevent both standard and non-protein aa mistranslation errors. We have identified a family of trans-editing factors collectively known as the INS superfamily, which ensures accurate and efficient translation of Pro and other codons. This family includes the cis-editing domain (INS) of bacterial prolyl-tRNA synthetase (ProRS), YbaK, and 4 ProXp's. While a subset of these proteins corrects ProRS-dependent errors, exciting preliminary studies have revealed distinct substrate specificities that extend beyond aa-tRNAPro and include non-protein aa's. The specific aims of this work are: 1) To determine the mechanism of substrate recognition by bacterial ProXp-ala; 2) To reveal the trans-editing function of bacterial ProXp-abu in vitro and in vivo; and 3) To probe the physiological roles of Escherichia coli INS superfamily members.